Assisting healing of skin-denuded areas on the human body with dried fibrous egg-shell membrane products and compositions therefor



United States Patent 0 3,196,675 ASSESTENG HEALWG 8F SKlJ-DENUDED AREAS ON THE HUMAN BGDY WEEK BREED FHERGUS EGG-SHELL MEMBRANE PRGBUCTS AND CUM- PQSETKONS THEREFQR Irene Neuhauser, 5759 53. Kenwood, Chicago, Ell. No Drawing. Filed Get. 3, 15369, Ser. No. 59,8 35 8 Claims. ill. 167-58) This application is a continuation-in-part of my application Serial No. 845,163, filed October S, 1959, now abandoned.

This invention is directed to new and useful methods for stimulating and assisting healing processes in damaged mammalian tissues as, for example, in areas of the human body where tissue has been lost or damaged as a result of injuries such as abrasions, cuts, burns, ulcerations and from surgical procedures and it is also concerned with the preparation of new and useful materials for effectively achieving these objectives.

Numerous approaches have heretofore been made and much study has been directed to problems involved in bringing about healing of areas where skin has been wholly or partially lost and where underlying tissues may have been damaged or removed as a result of burns, cuts, abrasions, ulcerations or from surgical intervention. The nature and character of the treatments currently used vary with techniques and preferences of individual surgeons and are dependent, also, on the nature, surface condition and extent and depth of tissue involvement.

A commonly employed procedure is that of viable skin grafting, in which the surgeon transplants shavings of living epidermal tissue from undamaged areas after suitable preparation. Such grafts have varied in size and thickness of the skin shavings used for grafting and in other details of procedure. Thiersch grafts, Ollier- Thiersch grafts, Wolfe grafts, pinch grafts, full-thickness and split-thickness grafts, and stamp grafts, are names associated with some of the various types or" skin grafts and procedures which have been used. In preparing skin shavings for grafts, the surgeon attempts to include portions of the stratum germinativum of the epidermis by cutting across the rete pegs. When such grafts become attached to the surface tissues of the damaged area, they may become vascularized and form islands or centers of epithelialization on the surface of the lesion. Grafts for which the patient is the donor of the grafted skin are known as autografts.

it frequently happens that the size of the lesion is such that not enough undamaged skin areas remain on the patient to provide autografts to cover the entire surface of the lesion. in such cases a portion of the lesion may receive an autograft and the remaining damaged area may receive grafts of living skin from another person, or of dead skin from a cadaver. Although such grafts, known as homografts, do not persist and do not become finally a part of the skin covering the healed lesion as autografts frequently do, they accomplish many desirable results such as preventing fluid loss, repressing the excessive development of granulation tissue, decreasing pain and stimulating growth of the stratum germinativum of the epidermis at borders of the lesion. Eventually homografts are rejected, that is, they slough olf. It may be possible by then to cut additional skin grafts from the now-healed donor sites and so eventually to cover the lesion completely with viable autografts. Considerable etfort has been directed to bring about the permanent survival of viable homografts but only slight pro ess has been achieved and no clinical applications of any of these studies have been widely accepted.

Although the homograft does not persist and become 3,l%,@75 Patented July 20, E965 a part of the skin over the healed lesion it is of great value as a practical measure since, by use of homografts together with autografts, lesions with greater areas and degrees of tissue damage may be satisfactorily treated. However, the limited availability of human skin in relation to the needs for horn-ografts makes it important that other more readily available materials, capable of performing one or more of the functions of the homograft, be found and that practical procedures for their preparation and use should be developed.

An area of approach which has been extensively studied, extending back approximately eighty years, has involved the utilization of egg-shell membranes, the membrana testacea of eggs of domestic fowl. Numerous workers have utilized egg-shell membrane as a subtitute for skin grafts but with varying degrees of success. In certain cases, where fresh egg-shell membrane was used in the repair of ruptured ear drums and as a graft to damaged areas from which skin had been entirely or partially lost, it was stated to have achieved reasonable success. In some cases, reported by other workers, fresh egg-shell membrane was found to be ineffective. Certain physiologists and surgeons concluded that satisfactory results could be obtained provided (1) that the egg-shell membrane pieces were applied with that surface towards the surface of the lesion which was previously in corn tact with the egg shell, and not the reverse, and (2) that the area was scarified until bleeding was produced prior to the application thereto of the eg -shell membrane. Unless t ese rules were carefully observed, according to certain of the workers in the art, the egg-shell membrane would not become attached organically to the denuded area to which it was applied. Another important criterion for the proper utilization of egg-shell membrane,

according to early workers in the art, was to insure that, when applied with the shell-side to the lesion, it be in contact with the proliferating cells of the stratum germinativurn of the skin at the periphery of the lesion. The said Workers reported that the egg-shell membrane itself, and of its own initiative, takes no part in the formation of connective tissue but that the eggshell membrane substance is transformed into connective tissue gradually, there being an interpenetration of the proliferating tissue into the structure of the membrane, followed by the establishment of circulation to care for the newly formed connective tissue structures.

Other workers have insisted that egg-shell membrane be applied with the albumen side toward the lesion, an exact reversal of the opinion of the above mentioned Workers. The basis for this was the belief that the inner side of the inner shell membrane contained nucleated cells which served as fuel for the healing processes. It is now known that such nucleated cells do not exist in eggshell membranes. Still other workers employed the shell membranes from boiled eggs but disregarded the question of which side was placed in contact with the lesion.

Despite repeated investigation of the possible use of eggshell membrane as a substitute for skin in grafting, the practice has not come into any appreciable or significant use. The difficulties of separating egg-shell membrane from eggs, the difiicult problems of handling moist membrane pieces and the indifferent success which has attended the use of egg-shell membrane as well as other factors, have all combined to make such approaches of dubious practical value.

In accordance with the present invention, highly important and significant improvements have been made which render the use of egg-shell membrane of great practical importance. It has been found, among other things, in accordance with the present invention, that egg- I adhesive materials.

shell membrane can be treated in such a manner to convert it into such forms as to make its use highly practicable and of great value in meeting problems encountered in connection with successful clinical mangement of skindenuded areas, deep-tissue wounds and lesions with are refractory to the usual treatment procedures. These have heretofore frequently required the utilization of auto grafts and to a lesser extent, and with less success, the use of homografts. The present invention makes possible the production of novel eg -shell membrane products, which are highly effective for use in place of human skin in skin grafting operations, which are easily manipulated in use and which can be made by simple practical procedures. Moreover, in the various forms in which the novel eggshell membrane products are produced, in accordance with the present invention, depending on the type and condition of the lesion to which they may be applied, they frequently display additional desirable properties such as a strong hemostatic action which tends to arrest bleeding, and a soothing, pain-relieving action, a type of action which some workers have reported to be a conspicuous property of homografts of human skin. Moreover, in the various forms in which the novel egg-shell membrane products are produced, in accordance with the present invention, they are readily and effectively storable so that they can be stockpiled in any desired amounts to meet emergency clinical situations which might be created due to holocausts, such as extended fires, wars and the like. These products constitute an effective treatment procedure providing a number of desirable characteristics and properties only some of which have heretofore been obtained by grafts of human skin.

The novel egg-shell membrane products made in accordance with the present invention play exceedingly important roles in tissue healing and grafting procedures. They serve, in fact, as effective biological dressings. In addition, by reason of their stimulating the regeneration of epithelial tissues and by providing topically certain factors which are essential aids in the natural processes of tissue repair, they appear actively to promote the regeneration of tissues by stimulation and possibly by providing essential components for synthesis of the protein of the regenerated tissues.

The present invention, in broad terms, involves the production and utilization or treatment of separated mainly fibrous particles prepared or derived from eg -shell membrane. Various effective procedures by which such may be accomplished are described in detail below.

Pursuant to one aspect of the present invention, eg shell membrane is converted into a fibrous pulp and the latter, in turn, is converted to egg-shell membrane felted fiber sheets, mats or non-woven fabrics. These sheets,

which may be made into any desired size or shape or after formation may be cut into any desired shape, may consist solely of egg shell membrane fibers or they may consist of mixtures of the egg-shell membrane fibers with various other materials. The egg-shell membrane fibers may, for example, constitute a coating or coatings on certain desirable base materials as, for instance, cellulose or alphacellulose or regenerated cellulose or cellulose acetate or nylon or similar natural or synthetic textile fabric or plastic base materials, woven or non-woven, in sheet form, perforated or imperforate, and, if desired, the egg-shell membrane fibers may be applied to either one or both sides of the base materials.

Furthermore, if desired, the egg-shell membrane fibers may be bonded to the base materials through suitable adhesive compositions such as compositions containing pectin, gelatin, starch and starch derivatives, innocuous vegetable gums, or like innocuous Typical of such felts or sheets are those made solely from uncooked eg -shell membrane fibers; those made solely from cooked egg-shell membrane fibers; those made from mixtures, in varying proportions, of uncooked egg-shell membrane fibers and cooked egg-shell membrane fibers; and those made from a mixture, for example, of uncooked egg-shell membrane fibers and 25% cotton fibers. Still others are in the form of composite sheets, one side comprising uncooked or cooked egg-shell membrane fibers and the other side comprising alpha-cellulose fiber pulp; and uncooked or cooked egg-shell membrane fibers deposited as an adherent or enmeshed coating or layer on a woven fabric such as surgical gauze.

In another aspect of my invention, instead of converting the egg-shell membrane fiber pulp into a dry felt or felted sheet form, as indicated above, the egg shell membrane fiber pulp may be dried to produce particulate solids of large surface area per unit of weight and used as such or, if desired, disaggregated into a finely divided fiuffy state.

In those instances where the eg -shell membrane fibers are mixed with other materials, for example, cellulosic materials such as alpha cellulose in fiber form, such latter materials should advantageously amount to not more than 75 and, better still, not greater than 25%, of the dry weight of the total of said material and said eg -shell membrane fibers. The cellulosic or like fibers or other materials may be admixed with the egg-shell membrane fiber pulp and the resulting fiber pulp mixture may then be laid down to form the felt or felted sheet or a coating or layer on a woven or non-woven base. Analogously, mixtures can be made where the egg-shell membrane fiber products are produced in the form of particulate solids, such masses having low bulk densities.

The egg-shell membrane fiber products with which the present invention is especially concerned are those which, unless converted by disaggregation or further disintegration steps as in the production of the dried, fluify particles hereafter described, are capable of being laid down, if desired, without addition of other fiber materials, to form dried felted fabrics or sheets which are in themselves form-sustaining and which can be handled reasonably without disintegration or breakage. They may, however, as described above, be admixed with other fibrous materials to produce composite felted fiber fabrics or sheets. In its broader aspects, the invention encompasses the production of separated egg-shell membrane fibers of such character as enable the preparation of such dried felted fiber fabrics or sheets and is not limited to the methods described above for the production of said separated eggshell membrane fibers or the dried, felted fiber fabrics or sheets produced therefrom. In the light of the teachings herein, other techniques of producing separated egg-shell membrane fibers effective for the production of formsustaining fiber fabrics or sheets will readily occur to those skilled in the art.

In the practice of the present invention, eggshell membrane is desirably washed and dried and then reduced to exceedingly small mainly fibrous particles. This is conveniently and very advantageously done in a high speed homogenizing apparatus. 1 have found that excellent results are obtained with a homogenizer such as a Vir Tis or the like, the cutting blades of which can be caused to rotate up to a speed of approximately 45,000 rpm. Other types of equipment can, however, be used. The usual Waring blenders and the Osterizers employed in the home for preparing dispersions and for disintegration and emulsification of food products have been found to give satisfactory results, especially if the rotating blades are first sharpened. Any other type of equipment may be employed so long as it and the details of procedure used serve .to convert the egg-shell membrane into finely divided mainly fibrous form. For example, dried eggshell membranes may be converted into finely divided particles having mainly a fibrous form by subjecting the dried egg-shell membranes to the action of a high speed homogenizer while the membrane pieces are suspended in a liquid which is readily imbibed by the dried egg-shell -membranes and thereby causes the dried membrane to amples or such liquids are water, aqueous solutions, metl yl alcohol, glacial acetic acid, molten phenol crystals, formamide, propionic acid, lactic acid, chlorophenols and cresols. Numerous other non-aqueous liquids which soften and swell dried egg-shell membrane can be used, those above mentioned being simply illustrative.

Generally speaking, the liquids which soften and swell the dried eggshell membranes and thereby condition the same for conversion by cutting, tearing or allied disintegrative action, as in an homogenizer, into a product comprising mainly separated egg-shell membrane fiber particles, are water-soluble or water-miscible and are of highly polar character. With some exceptions, of which glacial acetic acid is a particular example, these highly polar substances have a high dielectric constant. The highly polar liquids are imbibed by the dried egg-shell membrane and the extent to which such liquid is imbibed appears to be a measure of the extent of formation or the yield of separated egg-shell membrane er particles when homogenization or disintegration in the homogenizer is effected. Some liquids which are very poorly imbibed by dry egg-shell membranes are appreciably imbibed when a relatively small amount of water is present. It is to be noted that various of the non-aqueous materials can be used in the form of aqueous solutions, mixtures or suspensions. Another characteristic of many of the nonaqueous liquids which are effective softeners for dried eggshell membrane is that they have excellent hydrogen bonding tendencies. in the light of the foregoing teachings, it is apparent that numerous other non-aqueous liquids which are effective to bring about softening and swelling of dried egg-shell membranes with resultant conversion of the latter, of homogenization, mainly into separated fibers can readily be selected. it is also clear that the softening and swelling of dried egg-shell brane can be effected by subjection of the latter to vapors of certain of the softening and swelling agents. For instance, in the simplest case, dried eggshell membrane can be contacted with steam. i prefer, however, to utilize liquid media, and especially aqueous media, in the disintegration of egg-shell membranes into fibrous particles.

it has been found, in accordance with certain aspects of the present invention, that if the pieces of egg-shell membrane are disintegrated by high speed homogenizing or other disintegrative mechanical action, in an aqueous ambient or in a condition in which the membranes have imbibed water and become swollen and softened, as they are in the intact egg, the product which results is mainly fibrous in form and the individual fibers, in the moist condition, have a diameter which is fairly constant and is of the order of 4 microns, and usually of the order of 4- to 7 microns, when measured in the moist condition.

After homogenization or disintegration of the egg-shell membrane, the finely divided mainly fibrous particles can be readily separated from the suspending liquid employed. This step is accomplished by filtration, by centrifugation, by evaporation of the suspending liquid or by other Wellknown procedures for separating solids from liquids. The adhering residue of suspending liquid, if any, is then removed from the egg-shell membrane particles by Washing the particles with suitable solvents and these latter are removed by fil ration, centrifugation, evaporation or other convenient means. Care must be taken not to squeeze or compress the disintegrated or homogenized membrane mass as this tends to cause the formation of hard aggregates on drying. The resulting dry solid material consists of aggregies of small particles of disintegrated eggshell membrane. The dry aggregates of the mainly fibrous particles are easily disaggregated into separated mainly fibrous particles by acting on the above described aggregates in the homoge er in the absence of any suspending liquid, the egg: e pieces being disaggregated in air, or in other suitable gases, by high speed action of the homogenizer.

The dry, iluisy mass of egg-shell membrane fibers is now removed from th homogcnize The disaggregation of the dry mainly fibrous aggregates in the homogenizer or other suitable high speed disintegrator requires but a short period of action at high speeds. Prolonged subjection of the products to treatment in the disaggregating apparatus at high speeds may build up elevated temperatures in the product which could damage it.

Pursuant to one embodiment of the present invention, the mainly fibrous disaggregated disintegrated egg-shell membrane substance may be used for the therapeutic purpos s previously described.

Pursuant to another embodiment of my invention, the

above described mainly fibrous disintegrated disaggregated egg-shell membrane, as previously described, may be applied as a coating to cotton gauze, to paper, to woven and to non-woven fabrics, to plastic sheets both perforated and imperforate, or to other desirable base materials, on one or both sides, and the egg-shell membrane products may be made to adhere, if desired, to the surface of the base materials by means of suitable adhesive composiions, such as compositions containing pectin, gelatin, starch and/or starch derivatives, vegetable gums or soluble synthetic resinous polymeric substances and like innocuous adhesive materials.

in another important embodiment of my invention, as heretofore stated, a felt or felted sheet is formed in which the separated egg-shell membrane fibers mat together, or interlace, or become felted when these egg-shell membrane fibers, from boiled or unboiled egg-shell membranes, are laid down by procedures akin to those used in laying down felts of paper pulp, or sheets of paper, in paper making practice. To accomplish this aspect of the present invention, it is desirable that the separated eggshell membrane fibers contained in the disintegrated eggshell membrane material be of sufiicient average length to form a good felt or felted-fiber fabric or felted sheet from the suspension of egg-shell membrane fiber particles contained in the disintegrated egg-shell membrane suspension. In producing the aqueous suspension of eg -shell membrane fiber particles wherein equipment of the type of Vir Tis 45 homogenizer is employed, it should be noted that if the separated fibers are subjected too long to the action of the cutting blades of the Vir Tis 45 liomogenizer the fibers may become progressively shorter and there will be an increased proportion of exceedingly fine particles. It is distinctly preferred that the disintegrating, homogenizing or pulping step be carried out to reduce the egg-shell membrane material mainly to a suspension of fibers or fiber pulp in which the fibers of the egg-shell membrane material are predominantly in the free form. Excellent results are obtained where the individual fiber particles are mainly of a length within 20 to 600 microns with an average within the range 60 to 300 microns, and of an average thickness or diameter of the order of 4 to 7 microns when measured in the moist condi-tiol The egg-shell membranes which constitute the starting material used in carying out my invention can be separated from egg shells in any convenient manner. Thus, for example, this may be accomplished in a purely mechanical manner as, for instance, by rolling and pulling the membranes away from the washed egg shells after removal of the yolk and albumen of fresh or uncooked eggs. In the case of cooked eg -shell membranes, these, too, may be removed by pulling them away from washed egg-Shells which have been placed, for example, in boiling water to effect such cooking. In either case it is desirable to effect the separation of the egg-shell membranes from the egg-shells by grasping an edge of the membranes and rolling or pulling them away from the calcareous part of the egg shells.

Instead of employing purely mechanical means for effecting separation of the membranes from the egg shells, a combination of chemical and mechanical means can be utilized. One convenient procedure is to agitate,

in a churn or the like, water-washed, very coarsely chopped or shredded egg shells containing the adhering membranes with several times their weight of a dilute acid, for instance, a 0.25 to 4% solution in water of mineral acids such as hydrogen chloride, or a 2 to solution in water or organic acids such as formic acid, or acetic acid or lactic acid or propionic acid, for a period of time, generally one to several hours, agitating until the membranes are loosened and fall away from the egg shell particles. The membranes may then be separated from the egg shell pieces by counter-current Washing with water or aqueous solutions and by decantation steps. In case a small proportion of shell particles still remains attached to some membrane pieces, the membrane pieces are collected, washed with water by counter-current procedures and drained, then agitated for a period of one to four hours with a small amount of the dilute acid solution, followed by separation of the acid solution and counter-current washing of the membranes with decantation from bits of sedimented shell, using several changes of water.

The separation of the membrane from the shell may be effected with carbonic acid, that is, with an aqueous solution of carbon dioxide under pressure, though the process is time consuming. The time needed in this process may be decreased by agitating the shell pieces and by including with the shell material a cation exchange resin, for example, a cross-linked polystyrene polysulfonic acid, in the free acidic or hydrogen form, while the shell pieces are mixed with a solution of carbon dioxide in water, under compressed carbon dioxide. In these cases the separated membranes are finally washed with water or aqueous solutions using counter-current washing and decantation steps and the undissolved shell pieces and the cation exchange resin particles are separated with suitable sieves. The resin can then be regenerated to the free acid or hydrogen form by mineral acids, preferably hydrochloric acid, and is then ready for re-use.

In separating the membrane from the calcareous portions of the shell by means of dilute acids, separation is practically complete long before complete solution of the mineral part of the shell has taken place. It has been noted that the amount of mineral matter dissolved during the period necessary for membrane separation is much higher in the case of boiled egg shells than with uncooked egg shells, although the time of acid action is essentially the same. Evidently, boiling has created conditions in the shell which make more rapid the dissolving of the mineral matter by dilute acids.

The separated spent organic acid solutions may be regenerated for re-use conveniently by passing them through a column of appropriate cation exchange resin in acidic or hydrogen form and the cation exchange resins themselves may be regenerated to their acid forms with mineral acid solutions, such as dilute hydrochloric acid. Previous removal of the major portion of the dissolved calcium from'the spent acid by the addition of sulfuric acid in amounts just insufiicient to combine with all the calcium present and subsequent separation of the precipitated calcium sulfate by decantation and filtration, makes further regeneration of the dilute acid filtrate by treatment with cation exchange resins in hydrogen form a more eflicient and economical procedure.

Another suitable way of effecting separation of the membranes from the egg shells is illustrated by the following procedure. Approximately 37 grams of water-washed egg shells containing adhering membranes are chopped and placed in an acid resistant (for example, enameled) steel pressure vessel together with 1200 ml. of 6% w./v. acetic acid. The air in the vessel is displaced by carbon dioxide gas and then a pressure of 450 to 500 p.s.i. of carbon dioxide gas is maintained in said vessel for one hour. The carbon dioxide is then removed and suction by vacuum means is applied to the vessel. The suction is cut off, applied again, and again cut off. The vessel is then opened and the contents placed in a jar and agitated, any gases evolved being allowed to escape.

iembranes which separate from the egg shells are removed by decantation. Repeated washing with water is followed by decantations between washings which result in effecting removal of substantially all of the membranes from the egg shells and in removing all of the shell particles from the membranes.

The egg-shell membranes, after removal'from the egg shells, are desirably washed in Water or in aqueous media.

'For convenience in storage or subsequent handling, it

is desirable to subject them to a partial or complete drying operation depending on the physical form of the product desired from the subsequent homogenization or disintegration step. If the membranes are to be disintegrated, homogenized or pulped, for example, in an aqueous medium for the production chiefly of fibrous eggshell membrane particles, a partial removal of the Water contained in the Washed membranes may be accomplished simply by pressing the drained membranes between absorbent surfaces, for instance bibulous paper such as blotting pa er. In this form the blotter dry egg-shell membranes contain about 30% by Weight of membrane material, the balance being water, and they may be stored under refrigerated or freezing conditions for subsequent use in accordance with the teachings of my invention. Alternatively, the drained membranes may be dried in air or in a Vacuum drier and such dried membranes may be stored for prolonged periods, indeed almost indefinitely, at room temperatures without deterioration. They may be used in this form in the subsequent disintegration or homogenization step. However, in the case of dry membranes used in the disintegration or homogenization step in liquid media which are imbibed by the dry membrane and thereby soften the membrane, it is essential for best results to allow the dry membrane material to remain with the liquid medium a sufficient time to become saturated with the liquid and softened before commencing the mechanical disintegration, homogenization or pulping action of the homogenizer or other suitable equipment.

The following examples are illustrative of procedures which are useful in the production of egg-shell membrane mainly fibrous products pursuant to my invention. It will be understood that various changes can be made in the light of the teachings disclosed herein without departing from the essential guiding principles, and particularly with respect to translating the procedures to commercial scale operations.

Example 1 An approximate ratio of 5 grams of blotter dry mechanically separated egg-shell membranes (equal to about 1.5 grams air-dried membranes), which prior to blotter drying had previously been washed with water, are out up into thin strips about 71 inch to A inch wide and /4 to inch long and placed in the receptacle of an appropriate homogenizer, together with approximately ml. distilled water. The homogenizer is operated preliminarily (about 1 to 3 minutes) at low speed to break up the membranes and then is operated at full Speed (45,000 rpm.) for 13 minutes. The resulting fiber suspension is poured into a stainless steel screen box having a bottom made from stainless steel woven Wire screen having 200 openings per lineal inch. The box is held perfectly level and the Water is allowed to drain through the wire screen bottom. The resulting felted mass of eggshell membrane fiber is then sucked down with suction and an aluminum top plate is pressed down on the sheet to assist in removing the Water from on the sheet. After removing the top plate and then stopping the suction, 10 ml. of a 10% w./v. water solution of glycerol is carefully poured over all of the felted membrane fiber sheet, suction is then momentarily re-applied while the The top plate is pressed down on the sheet to force out as much liquid as possible. Suction is now interrupted and, while keeping the top plate in place, the screen box is placed in an inverted position on top of a conical receptacle so that the weight of the screen box and contents is supported by the pressure of the rim of the top of the receptacle against the top plate. The resulting set-up is allowed to stand overnight to allow evaporation of water through the screen interstices and the top plate is then removed leaving a dry sheet of felted egg-shell membrane fibers resting upon it and easily removable from it, having an area of 16 square inches.

The glycerol, which is desirably utilized, serves as a humectant so as to inhibit undue embrittlement or drying out of the felted sheet of eg -shell membrane fibers. In place of glycerol other hnmectants, generally aliphatic polyols, can be used, typical of which are sorbitol and propylene glycol. The humectant can be incorporated at any suitable stage of the process.

In the above example the eg -shell membrane fiber suspension in water may be mixed with a water suspension of finely divided fibers of cotton, of cellulose, of oxycellulose, of alpha cellulose, of regenerated cellulose, of silk, of rayon, of wool, or nylon and of other synthetic and natural fibers. The resulting sheet will be composite as to fiber types though each side of the sheet will be similar to the opposite side. Sheets with dissimilar sides may be prepared by developing on the screen box, in the manner described, first a felted sheet of one type of fiber and subsequently developing on top of the first sheet laid down, and while the latter is still in the screen box, a sec 0nd felted sheet from a dilferent fibrous material sus An approximate ratio of 1.5 grams of air-dried, mechanically separated, egg-shell membrane (equal to 5 grams blotter dry egg-shell membrane) together with approximately 170 ml. of distilled Water are placed in the receptacle of an appropriate homogenizer. After allowing the egg-shell membrane material to imbibe water so as to be in approximately equilibrium conditions with the water, the homogenizer is first operated at slow speed, as in Example 1, and then it is run at full speed for 13 minutes to convert the membrane into a fiber pulp. The resulting aqueous fiber pulp suspension is divided into two approximately equal portions each in a centrifuge bottle. The bottles so prepared are well shaken, then spun down for a period of about 10 minutes and the supernatant liquid is decanted. The residues are united in one bottle by washing one residue into the other bottle with 70 to 100 ml. of acetone, the latter is then shaken to suspend the residues, then spun down for about 10 minutes in the centrifuge and the liquid again decanted. The residue is then suspended in about 100 ml. acetone and collected on a Buchner suction funnel without using a filter paper. The acetone is allowed to drain away by gravity and the final residual acetone is allowed to evaporate spontaneously while preventing access of dust particles by wrapping the suction funnel in a cloth. The acetone-free, dry egg-shell membrane fiber aggregate is broken up by hand into small pieces, placed in the appropriate homogenizer in the receptacle without any liquid, and the homogenizer, after a preliminary operation of a few minutes at low speeds to break up the portions of egg-shell membrane fiber aggregate, is run at full speed for just a few seconds whereupon the egg-shell membrane fiber aggregate is completely disaggregated, and the resulting separated egg-shell membrane fibers are dispersed to produce a white, extremely light fluffy product, the bulk density of which is approximately 0.0195 grams per cc. In general, utilizing the foregoing type of procedure, the

bulk density of the product will usually fall Within the range of 0.01 to 0.08 gram per cc. Care must be exercised in this air-finding operation since, if the homogenizer is run for too long a period of time, the friction set up between the eggshell membrane and parts of the homogenizer may tend to cause some of the product to become scorched, giving an odor to the product. In general, treatment at high speed for approximately five to fifteen seconds is usually sufficient to achieve the desired results. Instead of carrying out the disaggregation or dispersion step in air, it can be done in any desired gaseous medium as, for instance, gaseous nitrogen, helium, argon, carbon dioxide and the like.

In place of acetone, other Water-miscible organic solvents having specific gravities preferably less than water can be employed, typical examples of which are methyl alcohol, ethyl alcohol and methyl-ethyl ketone. Various of such materials, in addition to functioning as dehydrating media and expediting the removal of moisture, also have a sterilizing effect on the egg-shell membrane fibers. Further, suspensions of egg-shell membrane fibrous material in such water-organic solvent media may be filtered instead of centrifuged and this is a convenient way to handle larger quantities of product.

Instead of removing the water or other suspending liquid from the egg-shell membrane fiber pulp suspension or the suspension of egg-shell membrane fibers resulting from the action of the homogenizer on the suspension of egg-shell membrane pieces, the water or other suspending liquid may be conveniently removed by subjecting the suspension of egg-shell membrane fibers to commercial drying, particularly spray-drying procedures, such as are used for the recovery of heat-labile solids from aqueous and other suspensions or solutions. membrane fiber aggregates resulting from the spray-drying operation may then be converted to an extremely light fluify product by subjecting these aggregates to the pre viously described disaggregative procedure for the preparation of the extremely light fluify product.

Example 3 This example was carried out in the manner described in Example 1 except that the starting egg-shell membranes were separated from the egg shells by the dilute acetic acid pressure procedure described above.

Example 4 This example was carried out in the manner described in Example 1 except that the fiber suspension, prior to being poured on the levelled stainless steel screen box, was admixed with alpha-cellulose fibers in an amount equal to about 25% of the dry weight of the egg-shell membrane solids. The resulting sheet consisted of an intimate mixture of the two fibrous materials and the two sides of the sheet were essentially similar.

Example 5 5 grams of blotter-dry eggshell membranes (such as described in Example 1) and 170 m1. of isopropyl alcohol were homogenized in the Vir Tis homogenizer operated at 45,000 rpm. for 13 minutes, using ice-bath cooling. Excellent disintegration was obtained. Examination under the micrometer microscope showed matted interlaced freed fibers and freed individual fibers. The longest fibers measured 485 microns and the shortest 18 microns. The average length was about 250 microns.

. Essentially all of the fibers were within the range of from 4 r to 7 microns in diameter.

This product is excellently adapted to making useful particulate products as well as sheet materials in the manners described above.

Example 6 The dry egg-shell fected at the start but after 1 minute an ice bath was put around the flask, more as a precaution against the formation of toluene vapors than for protection of the product. The egg-shell membranes rapidly disintegrated and after 12 minutes the machine was stopped. The disintegrated material was almost completely adherent to the walls of the flask. The toluene was decanted through a suction filter with a filter paper in place and the disintegrated product Was washed with acetone and collected on the filter paper. It was washed well with acetone and the acetone was then allowed to evapoarte in air at room temperature. Part of the disintegrated fairly fluily material coarsely chopped membranes were allowed to stand for a time in the suspending liquid before disintegration for periods up to several minutes to permit imbibition of liquid by the membrane to take place, and the egg-shell membrane-agent mixture was then subjected to the action of the homogenizer operating at a speed of 45000 r.p.m. Homogenization was carried out in the small receptacle using 80 ml. of liquid and the indicated quantity of airdried membrane. The temperature was controlled by a water bath or by an ice bath around the receptacle and the contents of the receptacle were thus kept from temperatures above C.

Suspending Liquid Weight of Air Dried Membrane Pieces (g.)

Time of Disintegratio Description of Product Formed membrane.

Melted Anhydrous Phenol.

action carried out at 45 C bibed.

imbibed by membrane.

bibed by membrane.

Mcrcapto-acetic Acid 98%.

Imbibed.

Mercapto-acetic Acid 80% in water. imbibed.

Formamide readily imbibed by Anhydrous Forrnic Acid readily Glacial Acetic Acid readily im- 0.75 Product mainly separated fibers. A few agglomerated or aggregated masses of separated fibers. No particles of eggshell membrane were seen. Fiber size: Longest 170 microns, Shortest 24 microns, Average 67 microns. Fiber diameter 3 to 7 microns when moist.

Almost entirely fibers, mostly iree individual fibers but some matted agglomerated or aggregated fibers. No pieces of original membrane. Fiber size in microns: Longest 218, shortest 37, Average length 60. Fiber diameter 3 to 7 when moist.

Product all fibers, no bits of original membrane. Mostly separated individual fibers but some agglomerated or aggregated mats. iber size in microns: Longest 290, shortest 30, Average 62. Fiber diameter 3 to 7 when moist.

Product entirely separated fibers with a few mats of agglomerated separated fibers. Fiber size in microns: Longest 387, Shortest 43, Average 100. Fiber diameter 3 to 7 when moist.

Product consisted almost completely of separated fibers. A few agglomerated mats of separated fibers. Fiber size in microns: Longest 265, Shortest 36, Average 84. Fiber diameter 3 to 7 when moist.

Product consisted almost entirely of separated fibers. A very few small agglomerations of these were seen. Fiber size in microns: Longest 290, Shortest 36, Average 90. Fiber diameter 3 to 7 when moist.

Product preponderantly separated fibers.

Remainder consists of agglomerated separated fibers, small particles and occasional small bits of membrane. Mats and pieces size in microns: 242 x 121, Smallest 90 x 50. Fiber size in microns: Longest 195, Shortest 22, Average 98. Fiber diameter 3 to 7 when moist.

was air dispersed or disaggregated in the Vir Tis homogenizer as described in Example 2 above. In such form it was excellently suitable as a particulate fiber material for topical treatment of wounds.

Another part of said disintegrated product was added to 170 ml. of distilled water, shaken well and a drop of the suspension was examined under the micrometer microscope. Examination showed three kinds of disintegrated products, (1) relatively large pieces, from 545 microns to 1815 microns largest dimension, (2) agglomerated mats of freed fibers, and (3) freed fibers. The fiber pieces in (2) and :(3) had fibers of about the same average length and were considerably longer to 545 microns) than the fibers ordinarily obtained in the usual aqueous ambient disintegration. I

The 170 ml. aqueous suspension was then converted into a 100% egg-shell membrane sheet in the manner described in Example 1. It gave an excellent sheet without the addition of any other fibrous material.

I have given the above several examples which will serve to illustrate various of the many types of procedure. It is to be understood that the invention itself is not limited thereby as additional procedures will be apparent to those skilled in the art.

The following table shows the results of several experi ments on egg-shell membrane disintegration utilizing various organic suspending liquids in the homogenizer. The

The egg-shell membrane products obtained from experiments 1 through 7 in the foregoing table can be effectively utilized in thepreparation of the fluffy fiber pulp form and they may also be used efiectively for the preparation of the sheet forms in the procedures as described above. Thus, for instance, the egg-shell membrane products shown in experiments 1 through 7 can be filtered 0%, and then (a) suspended in water and sheeted in the mannerdescribed herein, or (b) washed with acetone, air-dried and then disaggregated or flutied to produce a fluffy fiber pulp form of egg-shell membrane product having low bulk density.

The manner in which the dried egg-shell membrane particulate mainly fibrous materials of my invention are used to assist the healing of denuded areas on the human body is dependent, in part, on the nature of such areas. Thus, for example, if in the area the depth to which the tissue ha been lost is relatively superficial, a sheet of felted egg-shell membrane fibers or a sheet of other felted fibers admixed with fibrous egg-shell membrane products described above is simply placed over said area to cover same. To this end the sheet may be cut to conform in contour to that of the area to be covered. A protective cover of hospital gauze or the like may be laid gently and preferably relatively loosely over the sheet to protect the treated lesion and to prevent contamination.

If the denuded area is quite deep, as in the case of the surgical removal of an ulcer such as anecrotic ulcer, then arsaors the area is desirably packed with the dried egg-shell membrane mainly fibrous material. To this end, the 100% egg-shell membrane mainly fibrous material, if in sheet form, can be cut into small pieces and the latter packed or lightly tamped into the area throughout its depth. However, in this type of situation it is particularly desirable to pack the area with the dried egg-shell membrane material as produced in disaggregated particulate fibrous form. The area is again protected from contamination by hospital gauze as described above. As healing proceeds, additional amounts of the dried egg-shell membrane mainly fibrous materials may be applied to the denuded area as may be indicated by the rate and degree of healing.

The egg-shell membrane felted fiber fabrics or sheets and the egg-shell membrane particulate products produced in accordance with my present invention function, when utilized in the manner described above, as protective barriers for the denuded areas, exercising a mechanical protective action as well as markedly assisting in the healing of the lesion. The body fluids present in the tissues of the lesion may become intimately admixed with the particles of the egg-shell membrane mainly fibrous products and on drying form a reinforced crust having considerable mechanical protective strength. Further, the enzymic substances present in such body fluids and tissue exudates may act on the said egg-shell membrane particles, presenting as they do an extensive surface area for enzyme adsorption, and such enzymic action may result in the formation of substances which facilitate the healing process and/ or stimulate the growth of repair tissues and ultimately of new skin covering. The dried egg-shell membrane mainly fibrous products produced pursuant to my invention are characterized by exceptionally great surface area per unit of weight. This fact appears to play some role in relation to the manner in which said materials of my invention function in assisting the healing processes here involved, with due regard for the fact that the exact mechanism of the actions concerned in such processes is presently unknown.

It should be understood that, while it is preferred to utilize uncooked egg-shell membranes in the production or" the novel egg-shell membrane products of my invention, cooked or partially cooked egg-shell membranes can be treated in the same manners as described above in connection with the uncooked egg-shell membranes to produce the egg-shell membrane mainly fibrous products as well as to produce felts or felted fiber sheets or other essentially two-dimensional forms containing egg-shell membrane products, such as have been described herein above.

It has also been found that the egg-shell membrane fiber pulp prior to drying, or aqueous suspensions of the egg-shell membrane fibers, prepared as described above, may be treated in aqueous media with enzymes, preferably purified, of proteolytic character, such as papain, ficin, pepsin, trypsin and trypsin related enzymes, bro melin and other proteolytic enzymes of vegetable and/ or of animal origin so as to bring about partial digestion of the egg-shell membrane material. Moreover, the finished products may contain active proteolytic enzymes which serve, in certain instances, to enhance the utility of the egg-shell membrane digest product.

Useful products can also be prepared from the eggshell membrane mainly fibrous products herein described by oxidation and also by reduction techniques whereby to modify the disulfide linkages present in the egg-shell membrane substance. Oxidations have been carried out with hydrogen peroxide, urea peroxide, peracetic acid and performic acid to oxidize disulfide groups, present in eggshell membrane materials, to intermediate states of oxidation of the disulfide linkages and to form cysteic acid groups. Such latter acidic groups can be reacted chemically for instance with basic amino acids such as lysine, arginine, histidine, and with methionine, as well as with basic peptides. Reductions have been carried out .on the eg -shell membrane particulate mainly fibrous products described herein with mercaptoacetic acid, mercaptopropionic acid, substances which contain a reactive thiol group, and their soluble salts, to convert the disulfide groups present in the egg-shell membrane products to thiol groups. The resulting reduced egg-shell membrane products can then very readily be subjected to partial digestion with proteolytic enzymes to produce modified egg-shell membrane products useful for the treatment of denuded areas on the human body.

The dried eg -shell membrane mainly fibrous products, whether in sheet forms or in disaggregated particulate forms, can be sterilized, preferably prior to final packaging. This may be accomplished by techniques known in the art as, for example, by treatment with gaseous ethylene oxide propylene oxide, or mixtures of each of these gases with other gases such as mixtures of ethylene oxide gas with carbon dioxide gas, preferably under pressure and at lightly elevated temperatures, for example at 40 C. to C,, and at pressures of 2 to 6 atmospheres for a period of 2 to 4 hours. Sterilization may also be carried out using beta propiolactone or, in certain cases, with moist or dry heat. However, depending upon the exact manner in which the aforesaid egg-shell membrane products have been prepared and handled, they may have a practical degree of sterility without the necessity for any special sterilizing treatments.

Further, it may be advantageous under certain circumstances to combine the dried egg-shell membrane mainly fibrous products, whether in sheet forms or in disaggregated particulate forms, with drugs, with enzymes, with antibiotics and/or with bacteriostatic agents. To this end, said dried materials may be impregnated with the desired enzyme, drug, antibiotic or bacteriostatic substance or mixtures of these either by mechanical admixing of the said egg-shell membrane products with the drug in dry form or the drug may be dissolved in a suitable solvent and the drug solution applied to the eggshell membrane products at convenient points in the preparation of the egg-shell membrane products herein described or to the finished products themselves. Typical of such antibiotics are neomycin and bacitracin, and particularly, broad spectrum antibiotics such as chlortetracycline, tetracycline, oxytetracycline, chloramphenicol, the sulfa drugs and others.

While my invention is especially applicable to the treatment of egg-shell membranes obtained from chicken eggs, it is also useful in relation to egg-shell membranes obtained from eggs of other birds and fowl as, for instance, duck eggs. The examples listed above were carried out with egg-shell membranes prepared from the eggs of chickens because these represent the most available sources of large supplies of egg-shell membranes.

While I have described in detail processes of treating egg-shell membranes, it will be understood that other methods may be apparent to those skilled in the art.

What I claim as new and desire to protect by Letters Patent of the United States is:

1. As an article of manufacture for assisting the healing of skin-denuded areas on the body, dry separated eggshell membrane fibers in a finely divided form suitable for the preparation of form-sustaining egg-shell membrane dry felt sheets.

2. As an article of manufacture for assisting the healing of skin-denuded areas on the body, dry, finely divided flutfy particles of egg-shell membrane fibers the bulk density of said particles in the mass being from about 0.01 to 0.08 gram per cubic centimeter.

3. As an article of manufacture for assisting the healing of skin-denuded areas on the body, a mass of dry egg-shell membrane finely divided fibers, said fibers hav ing an average thickness within the range of 3 to 7 microns, when measured in the moist condition.

4. As an article of manufacture for assisting the healing of skin-denuded areas on the body, a mass of dry egg-shell membrane finely divided fibers, said fibers having a length mainly within the range of 20 to 500 mi 1 5 crons with an average length Within the range of 60 to 250 microns and having an average thickness Within the range of about 3 to 7 microns, when measured in the moist condition.

5. As an article of manufacture for assisting the healing of skin-denuded areas on the body, a dry felt sheet of finely divided egg-shell membrane fibers.

6. As an article of manufacture for assisting the healing of skin-denuded areas on the body, a dry sheet consisting of a layer of finely divided egg-shell membrane 1 .fibers on a fabric base member.

7. As an article of manufacture for assisting the healing of skin-denuded areas on the body, a dry felt sheet of a finely divided mixture of egg-shell membrane fibers with a non-cellulosic synthetic plastic material.

8. As an article of manufacture for assisting the healing of skin-denuded areas on the body, a dry felt sheet composed of finely divided egg-shell membrane fibers, the fibers having a length mainly within the range of 20 1.6 to 500 microns with an average length within the range of 60 to 250 microns, and an average thickness within the range of about 3 to 7 microns, When measured in the moist condition.

References Cited by the Examiner UNITED STATES PATENTS 2,039,312 5/36 Goldman 167-84 0 2,602,042 7/52 Abbott 167-65 2,833,669 5/58 Ziegler 167-84 OTHER REFERENCES Neuhauserz. A.M.A. Archives of Dermatology, vol. 75,

15 No. 3, pp. 401-404, March 1957.

JULIAN S. LEVITT, Primary Examiner.

WILLIAM B. KNIGHT, MORRIS O. WOLK, FRANK CACCIAPAGLIA, JR., LEWIS GOTTS, Examiners. 

1. AS AN ARTICLE OF MANUFACTURE FOR ASSISTING THE HEALING OF SKIN-DENUDED AREAS ON THE BODY, DRY SEPARATED EGGSHELL MEMBRANE FIBERS IN A FINELY DIVIDED FORM SUITABLE FOR THE PREPARATION OF FORM-SUSTAINING EGG-SHELL MEMBRANE DRY FELT SHEETS. 